PROJECT SUMMARY Kaposi's sarcoma-associated herpesvirus (KSHV), a member of the gamma-herpesvirus subfamily, has been shown to be an etiologic agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Kaposi's sarcoma is the most common malignancy associated with infection of human immunodeficiency virus (HIV) and develops in about 20% of acquired immunodeficiency syndrome (AIDS) patients without antiretroviral therapy. Viral latency of KSHV is thought to be directly linked to tumorigenesis, but viral lytic replication also contributes to tumorigenesis, directly and indirectly. Therefore, it is important to develop specific lytic replication inhibitors to control KSHV-associated cancers. Currently, the majority of the available drugs fighting herpesvirus infection are nucleic acid analogues targeting viral DNA synthesis. However, these nucleic acid drugs show extensive adverse effects including the frequent induction of drug resistant mutant viruses and various kinds of toxicity. Moreover, no drugs specifically targeting KSHV infection is available. Therefore, there is a need for the development of anti-KSHV drugs. We believe that aside from viral DNA synthesis, virus capsid assembly, which is a critical step for the production of progeny virions, can serve as a novel and potent drug target. KSHV capsids contain 955 capsid subunits. These subunits form a pressure-resistant network to withhold tens of atmospheres of pressure generated by its condensed ~150-kb dsDNA genome. We hypothesize that if an inhibitor binds to any one of the 955 capsid units, it will either prevent the formation of the capsid or result in an unstable capsid which bursts under the high pressure. The key point is that only one of the 955 units has to be targeted, thereby reducing the drug-to-target ratio by about a thousand-fold. Potentially, this method can reduce a drug's dose by ~ 3-Log, significantly minimizing its possible toxicity and side effects. In other words, the high internal pressure and large number of subunits make herpesvirus capsid assembly prone to interruption and can therefore be targeted for the structure-guided development of antiviral agents. Recently, we resolved the atomic-resolution structure of the KSHV capsid by employing electron-counting cryo- electron microscopy (cryoEM). Guided by the cryoEM structure and functional analysis results, we identified a promising druggable site, which is a hydrophobic groove on the upper-domain of Major Capsid Protein (MCP). The objective of this R21 application is to identify chemical inhibitors targeting this druggable site with the following specific aims: 1) to identify chemical inhibitors by two complementary high-throughput screenings, and 2) to identify lead compounds by functional evaluation. The lead compounds, which interrupt herpesvirus capsid assembly, will not only be a useful tool for scientific research but also have the potential to be the first- in-class drugs against herpesvirus infection.